Development and validation of an original magneto-chromatography device for the whole blood determination of hemozoin, the paramagnetic malaria pigment

Abstract

BackgroundMalaria remains the first global parasitic endemic disease with more than 400,000 deaths per year; there is a definite need for prevention measures and treatments, but also for rapid and low-cost diagnostic methods. Hemozoin, a detoxification polymer formed from heme by the parasite, and a likely biomarker of infection, has enticed many studies aiming at its whole blood determination. But, to our best knowledge, no accurate, precise and sensitive analytical method has been developed that could be implemented in endemic regions. ObjectivesOur group recently proposed a macroscopic trapping-dissolution method based on the paramagnetic properties of hemozoin crystals. The present paper further develops the concept into a workable fluidic device, validating an instrumental method that could be applied to the diagnosis of malaria. ResultsIn the newly developed integrated on-line system, the paramagnetic crystals are successively trapped through a superparamagnetic microbeads gradient field, dissolved by an alkaline solution, losing magnetic properties, eluted and quantified by spectrophotometry at 405 nm. The analysis time is comprised between 10 and 15 min. The performances of the method have been evaluated both on aqueous suspensions of β-hematin (a synthetic pigment with physical and paramagnetic properties analogous to those of hemozoin) and Plasmodium cultures, including the response function, linearity, precision, trueness, accuracy and quantification limits. From β-hematin suspensions and Plasmodium falciparum 3D7 cultures, the limit of detection would correspond to 80 and 55 parasites/µL whole blood, respectively (0.05 and 0.033 µg hemozoin/mL). ConclusionIn the absence of a reference method for the determination of hemozoin, its value as a malaria biomarker remains a matter of heavy debates. The newly developed magneto-chromatographic on-line system can discriminate the presence and absence of hemozoin in a sample but also accurately and precisely determine its level; application to whole-blood samples from strictly graded patients will allow to precise the usefulness of hemozoin for malaria diagnosis and/or prognosis.